HIV-1 particle production, a process driven by the viral structural protein Gag, takes place predominantly at the plasma membrane in cell types including T cells, whereas in macrophages a majority of virus particles appears to assemble in the late endosomes. The molecular mechanisms that determine the sites of virus assembly remain to be elucidated. The observed cell type difference is consistent with the possibility that a cellular factor(s) is involved in determining the subcellular location of HIV-1 assembly. We recently reported that depletion of the plasma membrane lipid PI(4,5)P2 in HeLa cells relocates Gag from the plasma membrane to late endosomes and markedly reduces virus particle production. Preliminary results show that Gag binding to total membrane is also impaired upon PI(4,5)P2 depletion. Recent structural data suggest that PI(4,5)P2 interacts directly with the matrix domain of Gag. These results suggest that a Gag-PI(4,5)P2 interaction is essential for virus assembly at the plasma membrane. Our goal in this proposal is to elucidate the molecular mechanisms by which PI(4,5)P2 regulates HIV-1 assembly in different cell types. Our specific aims are: [Aim 1] To identify Gag regions that are responsible for PI(4,5)P2 dependence. This will be achieved by a) characterization of Gag derivatives and b) isolation and characterization of viruses adapted to low PI(4,5)P2 levels. [Aim 2] To determine if PI(4,5)P2 regulates Gag localization by directly binding to Gag or through a PI(4,5)P2-dependent cellular trafficking pathway, or both. To address the first possibility, we will develop in vitro Gag-lipid binding assays. To examine the second possibility, we will analyze the impact of inhibition of membrane transport pathways and compare it with the effects of cellular PI(4,5)P2 depletion. [Aim 3] To examine, the role of PI(4,5)P2 in Gag localization to late endosomes and virus particle production in macrophages. We will perform quantitative analyses of PI(4,5)P2-Gag colocalization and virus release from macrophages with or without PI(4,5)P2 perturbation. The subcellular sites of virus assembly play key roles in the efficiency of virus production and viral persistence. Therefore, analyses of factors that determine the assembly sites have major implications in strategies for intervention in HIV-1 infection. The experiments proposed here will determine the roles played by PI(4,5)P2 in HIV-1 particle production and will contribute to the future development of antiviral drugs that target HIV-1 assembly and release.